Stem Cell Therapy Using Bone Marrow-Derived Muse Cells Repairs Radiation-Induced Intestinal Injury Through Their Intestine-Homing via Sphingosine Monophosphate-Sphingosine Monophosphate Receptor 2 Interaction

Purpose There is still no effective treatment for the gastrointestinal side effects of radiation therapy. Multilineage-differentiating stress-enduring (Muse) cells are tissue stem cells that have the ability to spontaneously home in on injured tissues and repair them. Several clinical trials have shown that stem cell therapy using human bone marrow-derived Muse (hBM-Muse) cells is effective in treating various diseases, but it is not known whether they are effective in treating radiation-induced intestinal injury. In this study, we investigated whether hBM-Muse cells are homing to the radiation-damaged intestine and promote its repair. Methods and Materials hBM-Muse cells were injected into the tail vein of mice 2 hours after high-dose total body irradiation. Then, homing analysis, crypt assay, bromodeoxyuridine assay, Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, immunostaining, and survival time measurements were performed. In addition, we analyzed the expression of sphingosine monophosphate (S1P), a Muse cell-inducing factor, in the mouse small intestine after irradiation. Finally, we investigated whether the administration of JTE-013, an S1P receptor 2-specific antagonist, inhibits hBM-Muse cells homing to the injured intestine. Results S1P expression increased in mouse intestine after irradiation, with hBM-Muse cells homing in on the injured intestine. Injection of hBM-Muse cells after radiation exposure significantly increased the number of crypts, proliferating cells in the crypts, and small intestinal component cells such as intestinal stem cells inhibited radiation-induced apoptosis and prolonged mouse survival. Treatment with JTE-013 significantly inhibited intestinal homing and therapeutic effects of hBM-Muse cells. These findings indicate that hBM-Muse cells homed in on the injured intestine through the S1P-S1P receptor 2 interaction to exert therapeutic effects on the radiation-induced intestinal injury. Conclusions This study indicates that hBM-Muse cells are effective in treating radiation-induced intestinal injury, suggesting that hBM-Muse cell-based stem cell therapy has the potential to overcome gastrointestinal side effects that limit the indications for radiation therapy.


Induction of differentiation of hBM-MSCs into adipocytes, osteocytes, and chondrocytes
Induction of hBM-MSC differentiation into adipocytes, osteocytes, and chondrocytes and detection of their respective differentiation markers was performed using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.Briefly, the cells were cultured for 2 to 3 weeks using the respective differentiation induction media included in the kit, and then FABP4 (adipocyte marker), Osteocalcin (osteocyte marker), and Aggrecan (chondrocyte marker) were detected by immunofluorescent staining.

Single-cell suspension culture, alkaline phosphatase (ALP) staining, differentiation into three germ layers
Single-cell suspension culture and ALP staining using hBM-Muse cells was performed as previously reported [1].Briefly, to avoid adhesion of hBM-Muse cells to the culture dishes, the dishes were coated with a 3% Poly (2-hydroxethyl methacrylate) (poly-HEMA; Sigma-Aldrich) solution in 95% ethanol.Isolated hBM-Muse cells were suspended in low-glucose DMEM (Thermo Fisher Scientific) containing 10% FBS (Serana Europe), 0.1 mg /ml kanamycin sulfate (Nacalai tesque) and 0.9% Methylcellulose (MethoCult H4100; StemCell Technologies, Vancouver, Canada), seeded onto poly-HEMA-coated culture dishes, and incubated for 5 days at 37℃ in 95% air and 5% CO2.MethoCult H4100 was used to avoid cell-to-cell adhesion between the hBM-Muse cells.ALP staining of hBM-Muse cell clusters at 5 days after single-cell suspension culture was performed using the Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich) according to the manufacturer's protocol.

Immunofluorescent staining
For immunofluorescent staining of tissues, tissue sections were deparaffinized, autoclaved in 10 mM citrate buffer at 120℃ for 15 min, and treated with 0.3% H2O2 to quench endogenous peroxidase activity.For immunofluorescent staining of cells, cells were fixed in 4% paraformaldehyde.Blocking and antibody dilution were performed using Blocking One Histo (Nacalai tesque) according to the manufacturer's protocol.Sections were incubated overnight at 4℃ with the primary antibodies listed in Table E2, and after washing, sections were stained with the appropriate fluorescence-conjugated secondary antibody for 1 hour at room temperature.Nuclei were stained using Hoechst dye (Thermo Fisher Scientific).Sections were visualized using an FV3000 (Olympus, Tokyo, Japan).

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay
Apoptosis was evaluated with paraffin-embedded sections of the jejunum by TUNEL assay using an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA) according to the manufacturer's protocol.Briefly, tissue sections were deparaffinized and treated with 20 μg/ml Proteinase K and 3% H2O2 in PBS.After equilibration with buffer, tissue sections were treated with a terminal deoxynucleotidyl transferase (TdT) enzyme to label the 3'-OH ends of DNA fragments with digoxigenin-nucleotides. Tissue sections were then incubated with a peroxidase-conjugated antidigoxigenin antibody at room temperature for 30 min and color development was performed using DAB as a chromogen.After staining, at least 10 different portions of the jejunum of each mouse were photographed under a microscope and the number of TUNEL+ cells in each crypt was assessed.In this study, TUNEL+ cells in approximately 200-250 crypts per mouse were measured.The average number of TUNEL+ cells/crypt for each group was obtained from four mice in each group.

Fluorescent labeling of cells
Fluorescent labeling of hBM-Muse or SSEA-3 -cells was performed using the LuminiCell Tracker 670-Cell Labeling Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol.Briefly, 400 µl 2 nM fluorescent labeling solution was added to 100 µl cell suspension (1 × 10 6 cells) and incubated at 37℃ in 95% air and 5% CO2, for 2 hours.The fluorescently labeled cells were then washed and used for injection into mice.In this Kit, the fluorescent dye enters the cell and the entire cell is labeled with intense fluorescence for a long term.

Measurement of S1P levels
After 10 Gy γ-ray TBI to BALB/c mice, the jejunum was removed at 24, 48, and 72 hours and stored frozen at -80℃.To examine the expression level of S1P in these tissues, LC-MS/MS analysis using TripleTOF 6600 (Sciex, Framingham, MA) and data analysis performed by Kazusa DNA Research Institute's Biomolecular Analysis Center (Chiba, Japan).The LC-MS/MS analysis in this study is a relative quantitative analysis.Three samples were analyzed from each group.E3.Then, membranes were washed and incubated with the appropriate HRPconjugated secondary antibody, washed, and developed with ECL Select reagents (GE Healthcare, Waukesha, WI).

Real-time PCR
Total RNA was isolated from cells and tissues using the RNeasy Mini Kit (QIAGEN, Venlo, Netherlands) and reverse transcribed using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) according to the manufacturer's protocol.Real-time PCR was performed on a LightCycler 480 (Roche Diagnostics) using the TaqMan probes (Thermo Fisher Scientific) listed in Table E4.The relative amount of each mRNA was normalized to the amount of Gapdh mRNA in the same sample.Real-time PCR analysis using the human ubiquitin (Ubc)-specific TaqMan probe (Hs00824723_m1) does not detect mouse Ubc (or other mouse genes).Therefore, to examine humanderived cells homing to the mouse small intestine, real-time PCR analysis using a human Ubc-specific TaqMan probe was performed.

RNA sequencing (RNA-seq)
RNA-seq library preparation, sequencing, mapping, and gene expression analysis were performed by DNAFORM (Kanagawa, Japan).Qualities of total RNA were assessed by Bioanalyzer (Agilent Technologies, Santa Clara, CA) to ensure a RIN (RNA integrity number) over 7.0.After poly (A) + RNA enrichment by NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Ipswich, MA), double-stranded cDNA libraries (RNA-seq libraries) were prepared using SMARTer Stranded Total RNA Sample Prep Kit HI Mammalian (Takara Bio, Shiga, Japan) according to the manufacturer's instruction.RNA-seq libraries were sequenced using paired end reads (50nt of read1 and 25nt of read2) on a NextSeq 500 instrument (Illumina, San Diego, CA).Obtained raw reads were trimmed and quality-filtered using the Trim Galore! (version 0.4.4),Trimmomatic (version 0.36), and cutadapt (version 1.16) software.Trimmed reads were then mapped to the human GRCh38.p13genome using STAR (version 2.7.2b).Reads on annotated genes were counted using featureCounts (version 1.6.1).Fragments Per Kilobase Million (FPKM) values were calculated from mapped reads by normalizing to total counts and transcript.E1

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Antibodies used for immunohistochemical staining

Table E2 .
Antibodies used for immunofluorescent staining

Table E3 .
Antibodies used for western blotting

Table E5 .
Other materials used in this study